Gonadotropin-releasing hormone (GnRH) is secreted in a pulsatile pattern from hypothalamic neurons to regulate gonadotropins in the anterior pituitary. As the ultimate regulator of reproductive function, GnRH is at the apex of the hypothalamic-pituitary-gonadal axis. It influences puberty, the menstrual cycle, fertility, menopause, and when disregulated, causes infertility. The overall goal of this renewal application is to elucidate the molecular mechanisms that regulate GnRH in hypothalamic neurons both at the level of gene expression and of pulsatile release. We will utilize two model systems: our immortalized GnRH-secreting hypothalamic cell line (GT1) and genetically manipulated mice. Expression of the GnRH gene is limited to approximately 800 hypothalamic neurons in the mouse. We have shown that a 300bp enhancer with a 173bp promoter target expression exclusively in cultured GT1 cells and transgenic mice. Furthermore, we have identified several homeodomain and other regulatory proteins that act through these regions to control GnRH expression. Remarkably, GT1 cells also release GnRH in a pulsatile fashion with a 30 minute periodicity mirroring mouse GnRH neurons in vivo. We propose three aims: Aim 1. We will investigate the mechanisms by which the neuron- specific enhancer and promoter target expression to the immortalized hypothalamic GnRH neurons. Our goals include identifying a GT1 cell-restricted protein that binds repeated elements to regulate the GnRH gene, understanding the mechanisms by which a complex of the homeodomain proteins Oct-1, Pbx, and Prep contributes to GnRH specificity, and determining the role of evolutionarily conserved elements in the enhancer and the far upstream region of the GnRH gene. Aim 2. Genetic manipulation of the mouse genome is a powerful approach to understanding the developmental and differentiated functions of GnRH neurons. Utilizing targeted recombination strategies, we will direct deletion of regulatory genes and dominant negative transcriptional regulators to GnRH neurons. We will also endeavor to develop a conditionally immortalized GnRH- expressing cell line by targeted oncogenesis in transgenic mice. Aim 3. The GT1 cell line exhibits pulsatile secretion of GnRH. Thus, the components of the pulse generator as well as the mechanisms for synchronization must be intrinsic to these immortalized cells. We plan to investigates the mechanisms of pulsatility utilizing long-term perifusion paradigms. Furthermore, the circadian clock is thought to set ultradian rhythms and mutant clock animals exhibit reproductive abnormalities. We will investigate the role of this transcriptional oscillator in mutant mice and in setting the pulse frequency of GnRH release in GT1 cells.